Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response

Abstract

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (T_FH) are considered important for mediating the antibody response. Most data supporting the role for T_FH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of T_FH-like cells in blood. We studied the activation and induction of T_FH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). T_FH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific T_FH by LAIV was studied by flow cytometry analysis of induced T_FH and CD154 expression. LAIV induced T_FH proliferation, which correlated with anti-HA antibody production, and T_FH were shown to be critical for the antibody response. Induction of T_FH from naive T cells by LAIV was shown in newly induced T_FH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced T_FH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced T_FH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific T_FH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific T_FH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, T_FH, are important for the antibody response. However, data on whether T_FH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing T_FH In this study, we demonstrate the induction of T_FH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that T_FH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections.

Keywords: LAIV; NALT; T follicular helper cell; TFH; antibody response; influenza vaccine; mucosal immunity; nasopharynx-associated lymphoid tissue.

FIG 1

LAIV induces T_FH proliferation that correlates with the GC B cell response and antibody production in NALT. LAIV stimulation induced increases in T_FH number (a and b) and T_FH proliferation (c and d) in tonsillar MNC (n = 15 for panels b and d; **, P < 0.01 versus unstimulated medium controls). (a and c) Representative plots and histogram for the T_FH subset (CXCR5^hi ICOS^hi) of CD4⁺ T cells following stimulation (day 3) (a) and for T_FH proliferation analyzed by CFSE staining (day 5) (red line, LAIV; gray shaded area, medium control) (c). (e and f) Increase in GC B cell number (CD19⁺ CD38^hi IgD⁻) in tonsillar MNC after LAIV stimulation (n = 13; **, P < 0.01 versus control). (g and h) LAIV-induced anti-HA IgG antibody production in tonsillar MNC (n = 20; ***, P < 0.01 versus control; day 8) (g) and LAIV-induced anti-HA IgG production in B cells cocultured with T_FH (red bars) or with non-T_FH cells (white bars) (n = 10; **, P < 0.01; #, P > 0.05 versus control) (h). Data in the bar figures are means and SE for a number of different experiments done with tonsils from different donors.

FIG 2

Induction of T_FH from naive tonsillar T cells and antibody production by LAIV. Representative plots (a) and a bar graph (b) show the induction of T_FH (CD4⁺ CXCR5⁺ ICOS⁺) from CD45RO⁻ MNC by LAIV compared to that for the medium control (n = 10; **, P < 0.01). (c and d) Fluorescence-activated cell sorting (FACS) histograms of BCL6 (c) and IL-21 (d) expression in LAIV-induced T_FH compared to that in unstimulated medium controls (isotype controls [gray shading]). (e) Dose-dependent induction of T_FH (day 7; top) and anti-HA IgG antibody production (day 14; bottom) from CD45RO⁻ MNC following LAIV stimulation (n = 6). (f) LAIV-induced anti-HA IgG, IgM, and IgA production in CD45RO⁻ MNC (day 14; n = 10; **, P < 0.01).

FIG 3

Detection of LAIV-induced antigen-specific T_FH and effects of IL-21, ICOS, CD40, and BCL6 signaling on T_FH and antibody induction. CD45RO⁻ MNC were first stimulated with LAIV for 7 days, followed by influenza virus antigen challenge with sH1N1 or HA antigen. (a) Representative plot showing activated T_FH (ICOS⁺ CXCR5⁺) following sH1N1 antigen challenge. (b) Representative plot showing the frequencies of activated T_FH (% of CD4⁺ T cells) after sH1N1 or HA challenge following prior LAIV stimulation (**, P < 0.01; ***, P < 0.001 versus LAIV stimulation alone). The medium-only negative-control level is also shown. (c and d) Representative plots (c) and frequency summary (n = 5) (d) for CD154 expression in the CD4⁺ T cell subsets, including T_FH, following sH1N1 antigen challenge. (e and f) Effects of neutralizing antibodies to IL-21R, ICOS-L, and CD40-L or a BCL6 blocker on T_FH induction (day 7) (e) and antibody production (day 14) (f) in CD45RO⁻ MNC following LAIV stimulation (**, P < 0.01 versus LAIV stimulation or use of isotype control antibodies).

FIG 4

IL-21 expression in LAIV-activated T_FH and its effect on anti-HA antibody production. (a) Representative plots showing T_FH subset and IL-21 expression levels in tonsillar CD4⁺ T cells following LAIV stimulation (including isotype control data [gray shading]). (b) Increase in IL-21-producing T_FH (% of CD4⁺ T cells) among tonsillar MNC following LAIV stimulation (n = 10; **, P < 0.01 versus control). (c to e) IL-21 concentrations following stimulation in the culture supernatants of tonsillar MNC (n = 22) (c), B cells cocultured with T_FH (n = 10) (d), or non-T_FH cells (n = 10) (e) (**, P < 0.01 versus control; NS, not significant). (f and g) IL-21R blocking by addition of anti-IL-21R antibody to tonsillar MNC led to reductions in T_FH number (f) and anti-HA IgG, IgM, and IgA antibody production (g) (n = 8; **, P < 0.01).

FIG 5

Activation of T_FH-like cells in PBMC. (a) Representative plots show the increase of T_FH-like cells (CD4⁺ CXCR5⁺ ICOS⁺) in PBMC following stimulation by LAIV for 3 days compared to their level in the medium control. (b) LAIV-induced increase in T_FH-like cells in PBMC compared to the control level (n = 10; **, P < 0.01). (c) Anti-HA IgG and IgM antibody production in PBMC culture supernatant following LAIV stimulation (n = 10; **, P < 0.01). (d) Frequency of antigen-specific CD154⁺ T_FH-like cells (% of CD4⁺ T cells; red bar) in PBMC following LAIV stimulation and subsequent sH1N1 antigen challenge compared to the frequencies of other CD4⁺ T cell subpopulations, as indicated (n = 4; **, P < 0.01; ***, P < 0.001).